PROJECT SUMMARY ? RP2: Emerging drug-resistant pathogens have outpaced drug discovery, which poses significant challenges for the development of safe and efficacious drugs. Our strategic approach addresses challenges in drug discovery by integrating human genetics, functional genomics, novel animal models, and innovative chemistry. Specifically, RP2 aims to develop host-directed therapeutics that harness innate intracellular defense mechanisms through induction of autophagy for the treatment of bacterial pathogens including S. Typhimurium, L. monocytogenes, S. aureus, multiple-drug-resistant strains thereof, and additional priority pathogens with RP1, RP3, and RP4. During the previous CETR funding period we have (1) completed 3 small molecule screens to identify autophagy-dependent anti-infective molecules, (2) completed several CRISPR screens to identify new targets controlling selective autophagy and lysosome homeostasis, and (3) leveraged human genetics and functional genomics to identify novel targets for therapeutic induction of antibacterial autophagy. Collectively, these studies advanced our objective of developing autophagy-directed therapeutics by generating novel lead compounds from phenotypic screens and precision targets from functional genomics. Moreover, our work has uncovered novel regulatory mechanisms governing autophagy and translated these discoveries to identify new points of entry for autophagy therapeutics. Our collaboration with RP1, RP3 and RP4 led to the discovery of small molecules that augment innate intracellular defense against diverse pathogenic microorganisms, including M. tuberculosis, S. flexneri, S. Typhimurium, arboviruses, norovirus, and T. gondii. In addition, we have partnered with Novartis to advance lead compounds directed at novel targets and to facilitate IND-enabling studies. In this CETR proposal, we will advance these autophagy-dependent anti- infective molecules using innovative chemistry (RP5) and validate new therapeutic targets from functional genomic and human genetic studies. We propose to leverage discoveries from the previous CETR Program to advance: Aim 1: medicinal chemistry to progress primary screen hits from three independent autophagy screens (LC3 puncta, NDP52-Salmonella co-localization and GPR65 agonist) to lead candidate autophagy-dependent broad-spectrum anti-infectives for in vitro and in vivo efficacy studies; Aim 2: development of targeting strategies to induce autophagy through TFEB, a master transcriptional regulator of autophagy and lysosome biogenesis genes; Aim 3: validation of novel genes identified from functional genomic and human genetic studies as therapeutic targets for antibacterial autophagy; and Aim 4: validation of novel anti-infective candidates generated by RP1-RP5 as inducers of anti-bacterial autophagy using in vitro and in vivo models. Together, our CETR team and industry partners are uniquely positioned to rapidly advance new treatments for emerging pathogens and infectious diseases.